Th1-, CTL-, and NK cell-driven cell-mediated immunity is the most effective form of response against cancer. Based on the novel paradigm of polarization of dendritic cells (DCs), and on our recent observations that the presence of IFNalpha allows for the generation of type-1 polarized DCs (DC1s) in serum-free conditions, we have recently developed the first clinically-applicable protocol to generate DC1s. Such alphaDC1s are the first clinically-applicable type of DCs that combine all 3 properties important for their in vivo activity as inducers of anti-cancer immunity. These are: 1) fully mature status; 2) high migratory responsiveness to CCR7 ligation; and 3) high IL-12p70-producing function. alphaDC1s show selectively enhanced ability to induce type-1 anti-cancer responses, when compared to the current "gold standard" of clinically-used DCs. Peptide-pulsed alphaDC1s induce up to 40-fold higher numbers of CTLs against MART- 1, tyrosinase, and gp100, during in vitro sensitization of peripheral blood T cells from melanoma patients. They also induce strong antitumor activity of CD4+ Th1 cells and NK cells. We hypothesize that alphaDC1-based vaccines administered by intralymphatic route will be safe and will act as superior inducers of tumor-specific responses in alphaDC1- treated melanoma patients in a novel phase I clinical trial. We will pursue one Specific Aim: Specific Aim 1. Demonstrate the safety and immunologic effectiveness of alphaDC1-based vaccines in patients with stage III/IV melanoma. We will perform a randomized phase I clinical trial (UPCI 03-118; IND#: 11,754) involving 28 patients, comparing the safety and the immunologic (and potentially clinical) efficacy of intralymphatic vaccination with aDC1s versus standard (IL-1beta/TNFalpha/IL-6/PGE2-matured) DCs. The immunomonitoring performed as a part of this aim will allow us to analyze the vaccination induced immunologic changes of melanoma-specific responses in circulation and DTH biopsies. The positive outcome of this study will allow us to obtain a proof of principle for the in vivo immunologic effectiveness of vaccines utilizing the phenomenon of DC polarization, and will help us to design an extended follow-up study of the immunologic and clinical effectiveness of alphaDC1-based vaccines. Verification of the in vivo efficacy of alphaDC1s, will also lead to follow-up studies of the mechanism of high activity of these cells in the induction of type-1 anticancer immunity and the prospective identification of the key mediators involved.